Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls

The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fertilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progression of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine content, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phenotypes. Bos indicus sperm samples (n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay (SCSA) and an optimized sperm protamine deficiency assay (SPDA). To verify the SPDA assay for bovine sperm protamine content, samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epididymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis (p < 0.01). The DNA fragmentation index (DFI), determined by SCSA, was positively correlated (r = 0.33 ± 0.08, p < 0.05) with the percentage of spermatozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated (r = -0.21 ± 0.09, p < 0.05) with the percentage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content and lower content of sperm DNA damage (p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull fertility. © 2014 American Society of Andrology and European Academy of Andrology.

Phyllosticta spp. on cultivated Citrus in Australia

The occurrence of pathogenic and endophytic species of Phyllosticta on cultivated Citrus in Australia was investigated by DNA sequence analysis of specimens held in plant pathology herbaria and culture collections. Sequences of the internal transcribed spacer region (ITS1, 5.8S, ITS2), and partial translation elongation factor 1-alpha (TEF) gene of 41 Phyllosticta-like isolates from Citrus were compared to those sequences from the type specimens of Phyllosticta recorded from around the world. Phylogenetic analysis resolved all the sequences of Australian accessions into two major clades. One clade corresponded to P. citricarpa, which causes citrus black spot disease. The other clade contained P. capitalensis, which is a known endophyte of Citrus and many other plant species. All included herbarium accessions previously designated as Guignardia mangiferae are now designated P. capitalensis. No Australian isolates were identified as the newly described pathogens of citrus P. citriasiana or P. citrichinaensis, or the endophytes Guignarida mangiferae, P. brazilianiae, or P. citribraziliensis. © 2013 Australasian Plant Pathology Society Inc.

Hybridisation, paternal leakage and mitochondrial DNA linearization in three anomalous fish (Scombridae)

Using mitochondrial DNA for species identification and population studies assumes that the genome is maternally inherited, circular, located in the cytoplasm and lacks recombination. This study explores the mitochondrial genomes of three anomalous mackerel. Complete mitochondrial genome sequencing plus nuclear microsatellite genotyping of these fish identified them as Scomberomorus munroi (spotted mackerel). Unlike normal S. munroi, these three fish also contained different linear, mitochondrial genomes of Scomberomorus semifasciatus (grey mackerel). The results are best explained by hybridisation, paternal leakage and mitochondrial DNA linearization. This unusual observation may provide an explanation for mtDNA outliers in animal population studies. © 2013.

Stretching single DNA-molecules with temperature-stabilized optical tweezers

This thesis consists of two parts; in the first part we performed a single-molecule force extension measurement with 10kb long DNA-molecules from phage-λ to validate the calibration and single-molecule capability of our optical tweezers instrument. Fitting the worm-like chain interpolation formula to the data revealed that ca. 71% of the DNA tethers featured a contour length within ±15% of the expected value (3.38 µm). Only 25% of the found DNA had a persistence length between 30 and 60 nm. The correct value should be within 40 to 60 nm. In the second part we designed and built a precise temperature controller to remove thermal fluctuations that cause drifting of the optical trap. The controller uses feed-forward and PID (proportional-integral-derivative) feedback to achieve 1.58 mK precision and 0.3 K absolute accuracy. During a 5 min test run it reduced drifting of the trap from 1.4 nm/min in open-loop to 0.6 nm/min in closed-loop.

Molecular phylogenetic analysis reveals six new species of Diaporthe from Australia

Six new species of Diaporthe, D. beilharziae on Indigofera australis, D. fraxini-angustifoliae on Fraxinus angustifolia subsp. oxycarpa, D. litchicola on Litchi chinensis, D. nothofagi on Nothofagus cunninghamii, D. pascoei on Persea americana and D. salicicola on Salix purpurea from Australia are described and illustrated based on morphological characteristics and molecular analyses. Three of the new species no longer produced sporulating structures in culture and two of these were morphologically described from voucher specimens. Phylogenetic relationships of the new species with other Diaporthe species are revealed by DNA sequence analyses based on the internal transcribed spacer (ITS) region, and partial regions of the β-tubulin (BT) and translation elongation factor 1-alpha (TEF). © 2013 Mushroom Research Foundation.

First report of Masseeëlla capparis in Australia

The rust fungus Masseeëlla capparis is reported for the first time in Australia on Flueggea virosa (Phyllanthaceae). This is the first species of Masseeëlla found in Australia. The rust is described and illustrated from Australian specimens. A discussion on species of Masseeëlla is provided. A reconstructed phylogeny with the Large Subunit region of ribosomal DNA indicated that Masseeëlla has an affinity with the Phakopsoraceae.

An improved real-time PCR assay for the detection of Old World screwworm flies

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. © 2014 International Atomic Energy Agency.

Rapid assessment of genotype-by-environment interactions and heritability for growth rate in aquaculture species using in vitro fertilisation and DNA tagging

Commercial environments may receive only a fraction of expected genetic gains for growth rate as predicted from the selection environment. This fraction is result of undesirable genotype-by-environment interactions (GxE) and measured by the genetic correlation (rg) of growth between environments. Rapid estimates of genetic correlation achieved in one generation are notoriously difficult to estimate with precision. A new design is proposed where genetic correlations can be estimated by utilising artificial mating from cryopreserved semen and unfertilised eggs stripped from a single female. We compare a traditional phenotype analysis of growth to a threshold model where only the largest fish are genotyped for sire identification. The threshold model was robust to differences in family mortality differing up to 30%. The design is unique as it negates potential re-ranking of families caused by an interaction between common maternal environmental effects and growing environment. The design is suitable for rapid assessment of GxE over one generation with a true 0.70 genetic correlation yielding standard errors as low as 0.07. Different design scenarios were tested for bias and accuracy with a range of heritability values, number of half-sib families created, number of progeny within each full-sib family, number of fish genotyped, number of fish stocked, differing family survival rates and at various simulated genetic correlation levels.

Plasmopara sphagneticolae sp. nov. (Peronosporales) on Sphagneticola (Asteraceae) in Australia

A specimen of downy mildew on leaves of Sphagneticola trilobata found in northern Queensland was identified by a systematic approach as a novel species of Plasmopara. A new species, Plasmopara sphagneticolae, is proposed for this specimen, which differs from other species of Plasmopara by morphology, host range, and sequence data from nuclear-ribosomal DNA and mitochondrial DNA. Plasmopara sphagneticolae, together with P. halstedii, are downy mildews found on host species in the tribe Heliantheae (Asteraceae). Plasmopara halstedii causes downy mildew on Helianthus annuus, and is not present on sunflower in Australia. Phylogenetic analysis of the large subunit region of ribosomal DNA showed that P. sphagneticolae was sister to P. halstedii on sunflower.

Uromycladium falcatarium sp. nov., the cause of gall rust on Paraserianthes falcataria in south-east Asia

The gall rusts on Acacia spp. and Paraserianthes falcataria are caused by species of Uromycladium. Morphology and a phylogenetic analysis of four loci from ribosomal (SSU, ITS, LSU) and mitochondrial (CO3) DNA, showed that the rust on P. falcataria differed from U. tepperianum. Uromycladium falcatarium sp. nov. is described to accommodate this taxon, which can be differentiated from other species of Uromycladium by teliospore wall morphology, host genus and DNA sequence data.

Three new species of Tilletia on Eriachne from north-western Australia

Three new species of Tilletia are described from species of Eriachne (Poaceae) in the arid tropics of north-western Australia. In Western Australia, T. mactaggartii sp. nov. infects E. burkittii, and T. geeringii sp. nov. infects E. festucacea. Tilletia marjaniae sp. nov. infects E. pulchella subsp. dominii in Western Australia and the Northern Territory. These species are the first records of Tilletia on Eriachne. Phylogenetic relationships of these species were inferred from internal transcribed spacer of ribosomal RNA region and large subunit ribosomal RNA gene sequences.

Polyphasic characterization of four new plant pathogenic Phyllosticta species from China, Japan, and the United States

The black rot disease of Vitis species and other host genera of Vitacease is caused by Phyllosticta ampelicida and allied taxa which is considered to be a species complex. In this paper, we introduce four new species of Phyllosticta, including two from the P. ampelicida complex, based on a polyphasic characterization including disease symptoms and host association, morphology, and molecular phylogeny. The phylogenetic analysis was conducted based on the ribosomal internal transcribed spacer (ITS) region and a combined multi-locus alignment of the ITS, actin (ACT), partial translation elongation factor 1-alpha (TEF-1), and glyceraldehydes 3-phosphate dehydrogenase (GPDH) gene regions. Our study confirms the phylogenetic distinctions of the four new species, as well as their phenotypic differences with known species in the genus.

A retrospective study of Babesia macropus associated with morbidity and mortality in eastern grey kangaroos (Macropus giganteus) and agile wallabies (Macropus agilis)

This is a retrospective study of 38 cases of infection by Babesia macropus, associated with a syndrome of anaemia and debility in hand-reared or free-ranging juvenile eastern grey kangaroos (Macropus giganteus) from coastal New South Wales and south-eastern Queensland between 1995 and 2013. Infection with B. macropus is recorded for the first time in agile wallabies (Macropus agilis) from far north Queensland. Animals in which B. macropus infection was considered to be the primary cause of morbidity had marked anaemia, lethargy and neurological signs, and often died. In these cases, parasitised erythrocytes were few or undetectable in peripheral blood samples but were sequestered in large numbers within small vessels of visceral organs, particularly in the kidney and brain, associated with distinctive clusters of extraerythrocytic organisms. Initial identification of this piroplasm in peripheral blood smears and in tissue impression smears and histological sections was confirmed using transmission electron microscopy and molecular analysis. Samples of kidney, brain or blood were tested using PCR and DNA sequencing of the 18S ribosomal RNA and heat shock protein 70 gene using primers specific for piroplasms. The piroplasm detected in these samples had 100 sequence identity in the 18S rRNA region with the recently described Babesia macropus in two eastern grey kangaroos from New South Wales and Queensland, and a high degree of similarity to an unnamed Babesia sp. recently detected in three woylies (Bettongia penicillata ogilbyi) in Western Australia.

Studies on CNTs/DNA composite

The electrical and optical properties of MWCNTs/DNA composite were studied. Electrical conductivity studies reveal that, the increase in CNTs concentration in DNA increases the conductivity. Fourier transformed Infrared (FTIR) spectrum shows that the CNTs are bonded to DNA covalently at the ends and defects sites and the wrapping of DNA on the CNTs is due to van der Waals force.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to a canonical LAGLIDADG homing endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches including four complementary footprinting assays such as DNase I, Cu/phenanthroline, methylation protection and KMnO4, enhancement of 2-aminopurine fluorescence and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site and generates two staggered double-strand breaks. Taken together, these results implicate that PI-MleI possess a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of LAGLIDADG family of homing endonucleases